EP1005532B1 - Isolierung und verwendung von nukleinsäuremolekülen, welche für mitglieder der ssx-familie kodieren - Google Patents

Isolierung und verwendung von nukleinsäuremolekülen, welche für mitglieder der ssx-familie kodieren Download PDF

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Publication number
EP1005532B1
EP1005532B1 EP98910056A EP98910056A EP1005532B1 EP 1005532 B1 EP1005532 B1 EP 1005532B1 EP 98910056 A EP98910056 A EP 98910056A EP 98910056 A EP98910056 A EP 98910056A EP 1005532 B1 EP1005532 B1 EP 1005532B1
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EP
European Patent Office
Prior art keywords
seq
nucleic acid
isolated nucleic
acid molecule
sample
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EP98910056A
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English (en)
French (fr)
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EP1005532A1 (de
EP1005532A4 (de
Inventor
Ali O. Gure
Ozlem Tureci
Ugur Sahin
Solam Tsang
Matthew J. Scanlan
Alexander Knuth
Michael Pfreundschuh
Lloyd J. Old
Yao-Tseng Chen
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Ludwig Institute for Cancer Research Ltd
Cornell Research Foundation Inc
Memorial Sloan Kettering Cancer Center
Ludwig Cancer Research
Original Assignee
Ludwig Institute for Cancer Research Ltd
Cornell Research Foundation Inc
Memorial Sloan Kettering Cancer Center
Ludwig Cancer Research
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Priority to EP02024769A priority Critical patent/EP1300463B1/de
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Publication of EP1005532A4 publication Critical patent/EP1005532A4/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • This invention relates to the isolation and cloning of genes which are members of the "SSX" family, which is discussed herein, and the uses thereof.
  • pathological conditions such as infections, cancer, autoimmune disorders, etc.
  • these molecules thus serve as "markers” for a particular pathological or abnormal condition.
  • diagnostic "targets” i.e., materials to be identified to diagnose these abnormal conditions
  • the molecules serve as reagents which can be used to generate diagnostic and/or therapeutic agents.
  • a by no means limiting example of this is the use of cancer markers to produce antibodies specific to a particular marker.
  • Yet another.non-limiting example is the use of a peptide which complexes with an MHC molecule, to generate cytolytic T cells against abnormal cells.
  • the biochemical approach exemplified by, e.g., Mandelboim, et al., Nature 369: 69 (1994) is based on acidic elution of peptides which have bound to MHC-class I molecules of tumor cells, followed by reversed-phase high performance liquid chromography (HPLC).
  • Antigenic peptides are identified after they bind to empty MHC-class I molecules of mutant cell lines, defective in antigen processing, and induce specific reactions with cytotoxic T-lymphocytes. These reactions include induction of CTL proliferation, TNF release, and lysis of target cells, measurable in an MTT assay, or a 51 Cr release assay.
  • the methodologies described rely on the availability of established, permanent cell lines of the cancer type under consideration. It is very difficult to establish cell lines from certain cancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg. Clin. North. Am. 10: 607-637 (1990). It is also known that some epithelial cell type cancers are poorly susceptible to CTLs in vitro, precluding routine analysis. These problems have stimulated the art to develop additional methodologies for identifying cancer associated antigens.
  • SSX2 a triad of genes
  • HOM-MEL-40 tumor associated antigen referred to as HOM-MEL-40 by Tureci, et al, supra . Its expression to date has been observed in cancer cells, and normal testis only. Thus parallels other members of the "CT" family of tumor antigens, since they are expressed only in cancer and testis cells. Crew et al. also isolated and cloned the SSX1 gene, which has 89% nucleotide sequence homology with SSX2. See Crew et al., supra . Additional work directed to the identification of SSX genes has resulted in the identification of SSX3, as is described by DeLeeuw, et al., Cytogenet. Genet 73:179-183 (1996). The fact that SSX presentation parallels other, CT antigens suggested to the inventors that other SSX genes might be isolated.
  • a human testicular cDNA expression library was obtained, and screened, with serum from a melanoma patient identified as MZ2. See e.g., parent application U.S. Patent No. 5,698,396; also see U.S. Patent No. 5,804,381; Sahin, et al., Proc. Natl. Acad. Sci. USA 92:11810-11813 (1995). This serum had been treated using the methodology described in these references. Briefly, serum was diluted 1:10, and then preabsorbed with transfected E. coli lysate. Following this preabsorption step, the absorbed serum was diluted 1:10, for a final dilution of 1:100.
  • nitrocellulose membranes containing phage plaques prepared using the methodology referred to supra .
  • the nitrocellulose membranes were washed, incubated with alkaline phosphatase conjugated goat anti-human Fc ⁇ secondary antibodies, and the reaction was observed with the substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium.
  • any phagemids which encoded human immunoglobulin were eliminated.
  • autoimmune disease associated molecules i.e., Golgin - 95 (Fritzler, et al., J. Exp. Med. 178:49-62 (1993)), and human upstream binding factor (Chan, et al., J. Exp. Med. 174:1239-1244 (1991)).
  • Three other clones were found to encode for proteins which are widely expressed in human tissue, i.e., ribosomal receptor, collagen type VI globular domain, and rapamycin binding protein.
  • RT-PCR methodology described supra was carried out on testicular total RNA, and the amplification product was used in southern blotting experiments.
  • Genomic DNA was extracted from non-neoplastic tissue samples, and then subjected to restriction enzyme digestion, using BamHI, Eco RI, or HindIII in separate experiments and then separated on a 0.7% agarose gel, followed by blotting on to nitrocellulose filters.
  • the amplification products described supra were labeled with 32 P, using well-known methods, and the labeled materials were then used as probes under high stringency conditions (65°C, aqueous buffer), followed by high stringency washes, ending with a final wash at 0.2xSSC, 0.2% SDS, 65°C.
  • SSX1, 2 and 3 An analysis of the sequences of SSX1, 2 and 3 revealed that SSX1 and 2 contained a BglII site which was not shared by SSX3. Similarly, SSX3 contained an EcoRV site not shared by the other genes.
  • testicular cDNA was amplified, using SEQ ID NOS: 3 and 4, as described supra, and was then subjected to BglII digestion. Any BglII resistant sequences were then cloned, sequenced, and compared with the known sequences.
  • SSX4 a previously unidentified sequence, referred to hereafter as SEQ ID NO: 1 herein.
  • a search of the GenBank database found two clones, identified by Accession Number N24445 and W00507, both of which consisted of a sequence - tag - derived cDNA segments.
  • the clone identified by N24445 contained the 3'-untranslated region of SSX4, and part of its coding sequence, while the one identified, as W00507 contained a shorter fragment of the 3'-untranslated region of SSX4, and a longer part of the coding sequence.
  • N24445 consists of base 344 of SSX4 (SEQ ID NO:1), through the 3-end, plus 319 bases 3' of the stop codon.
  • the W00507 sequence consists of a 99 base pair sequence, showing no homology to SSX genes followed by a region identical to nucleotides 280 through the end of SEQ ID NO:1, through 67 bases 3' of the stop codon of SEQ ID NO:1.
  • SEQ ID NO: 1 Two forms of SSX4 (SEQ ID NO: 1) were identified. One of these lacked nucleotides 331 to 466 but was otherwise identical to SSX4 as presented in SEQ ID NO: 1. As is described infra, the shorter form is an alternatively spliced variant.
  • Table 1 which follows, the nucleotide and amino acid sequences of the 5 known members of the SSX family are compared. One reads the table horizontally for nucleotide homology, and vertically for amino acid homology. Hence, SSX1 and SSX4 share 89.4% homology on the nucleotide level, and 79.3% homology on the amino acid level.
  • SSX4 When the truncated form of SSX4 is analyzed, it has an amino acid sequence completely different from others, due to alternate splicing and shifting of a downstream open reading frame.
  • the putative protein is 153 amino acids long, and the 42 carboxy terminal amino acids show no homology to the other SSX proteins.
  • genomic human placental library in lambda phage was screened, using the same protocol and probes described supra in the discussion of the southern blotting work: Any positive primary clones were purified, via two additional rounds of cloning.
  • SSX2 The analysis revealed that the SSX2, gene contains six exons, and spans at least 8 kilobases. All defined boundaries were found to observe the consensus sequence of exon/intron junctions, i.e. GT/AG.
  • SEQ ID NOS: 5-14 The specificity of the clones was confirmed by amplifying, the previously identified cDNA for SSX1 through SSX5. Taq polymerase was used, at 60°C for SSX1 and 4, and 65°C for SSX2, 3 and 5. Each set of primer pairs was found to be specific, except that the SSX2 primers were found to amplify minute (less than 1/20 of SSX2) amounts of SSX3 plasmid DNA.
  • the primers were used to analyze testicular mRNA, using the RT-PCR protocols set forth supra.
  • SSX genes in cultured melanocytes were then studied. RT-PCR was carried out, using the protocols set forth supra . No PCR product was found. Reamplification resulted in a small amount of SSX4 product, including both alternate forms, indicating that SSX4 expression in cultured melanocytes is inconsistent and is at very low levels when it occurs.
  • the isolated nucleic acid molecules of the invention encompass those degenerate sequences which, though not identical to SEQ ID NO: 1 or its splice variant, do encode the same proteins which these sequences encode. Also a part of the invention are expression vectors which comprise these molecules, operably linked to a promoter, and the cell lines or cell strains which are transformed or transfected with these vectors, or the nucleic acid molecules themselves. These are all useful in making the protein, e.g., as well as for producing amplified copies of the relevant sequences.
  • nucleic acid molecules defined herein by SEQ ID NOS: 11 and 12 are those nucleic acid molecules defined herein by SEQ ID NOS: 11 and 12, compositions containing these, and the use thereof in assaying for expression of one or more SSX gene.
  • Any nucleic acid hybridization methodology can be used, including, e.g., PCR methodologies.
  • the antibodies of the invention may also be used in assays, but in this case the target is the expression product of the SSX genes.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Claims (14)

  1. Isoliertes Nucleinsäuremolekül, das für ein Protein kodiert, dessen Aminosäuresequenz aus der Aminosäuresequenz besteht, für die Seq.-ID Nr. 1 kodiert oder die Nucleotide 1-330, verkettet mit den Nucleotiden 467-576 von Seq.-ID Nr. 1 kodieren.
  2. Isoliertes Nucleinsäuremolekül nach Anspruch 1, worin das isolierte Nucleinsäuremolekül für das Protein kodiert, für das Seq.-ID Nr. 1 kodiert.
  3. Isoliertes Nucleinsäuremolekül nach Anspruch 1, worin das isolierte Nucleinsäuremolekül für das Protein kodiert, für das die Nucleotide 1-330, verkettet mit den Nucleotiden 467-576 von Seq.-ID Nr. 1 kodieren.
  4. Isoliertes Nucleinsäuremolekül nach Anspruch 1, das aus der Nucleotidsequenz von Seq.-ID Nr. 1 besteht.
  5. Isoliertes Nucleinsäuremolekül nach Anspruch 1, das aus der Nucleotidsequenz besteht, die durch die Nucleotide 1-330, verkettet mit den Nucleotiden 467-576, wie in Seq.-ID Nr. 1 angegeben, definiert wird.
  6. Expressionsvektor, der ein isoliertes Nucleinsäuremolekül nach einem der Ansprüche 1 bis 5 mit einem Promotor operabel verbunden umfasst.
  7. Zelllinie oder Zellstamm, die bzw. der mit einem isolierten Nucleinsäuremolekül nach einem der Ansprüche 1 bis 5 transformiert oder transfiziert ist.
  8. Zelllinie oder Zellstamm, die bzw. der mit einem Expressionsvektor nach Anspruch 6 transformiert oder transfiziert ist.
  9. Isoliertes Protein, für das ein isoliertes Nucleinsäuremolekül nach einem der Ansprüche 1 bis 5 kodiert.
  10. Isoliertes Nucleinsäuremolekül, das zum Bestimmen der Expression eines SSX4-Gens in einer Probe nützlich ist, wobei das isolierte Nucleinsäuremolekül aus der in Seq.-ID Nr. 11 oder Seq.-ID Nr. 12 angegebenen Nucleotidsequenz besteht, wie in Tabelle 2 gezeigt.
  11. Zum Bestimmen der Expression eines SSX-Gens in einer Probe nützliche Zusammensetzung, umfassend (a) Seq.-ID Nr. 3 und Seq.-ID Nr. 4, wie in Beispiel 2 gezeigt, (b) Seq.-ID Nr. 5 und Seq.-ID Nr. 6, (c) Seq.-ID Nr. 7 und Seq.-ID Nr. 8, (d) Seq.-ID Nr. 9 und Seq.-ID Nr. 10, (4) Seq.-ID Nr. 11 und Seq.-ID Nr. 12, und (f) Seq.-ID Nr. 13 und Seq.-ID Nr. 14, wie in Tabelle 2 gezeigt.
  12. Verfahren zum Bestimmen der Expression eines SSX4-Gens in einer Probe, umfassend das Kontaktieren der Probe mit zumindest einem isolierten Nucleinsäuremolekül nach Anspruch 10 und das Bestimmen der Hybridisierung des isolierten Nucleinsäuremoleküls an ein Target als Bestimmung der Expression eines SSX4-Gens in der Probe.
  13. Isolierter Antikörper, der sich spezifisch an ein isoliertes Protein nach Anspruch 9 bindet.
  14. Verfahren zum Nachweis der Gegenwart eines Expressionsprodukts eines SSX4-Gens in einer Probe, umfassend das Kontaktieren der Probe mit einem isolierten Antikörper nach Anspruch 13 und das Bestimmen der Bindung des Antikörpers an ein Target als Nachweis der Gegenwart des Expressionsprodukts eines SSX4-Gens in der Probe.
EP98910056A 1997-05-05 1998-02-25 Isolierung und verwendung von nukleinsäuremolekülen, welche für mitglieder der ssx-familie kodieren Expired - Lifetime EP1005532B1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP02024769A EP1300463B1 (de) 1997-05-05 1998-02-25 Isolierung und Verwendung von Nukleinsäuremolekülen, welche für Mitglieder der SSX-Familie kodieren

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US08/851,138 US6291658B1 (en) 1997-05-05 1997-05-05 Isolated nucleic acid molecules encoding SSX family members and thereof
US851138 1997-05-05
PCT/US1998/003661 WO1998050528A1 (en) 1997-05-05 1998-02-25 Isolated nucleic acid molecules encoding ssx family members and uses thereof

Related Child Applications (1)

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EP02024769A Division EP1300463B1 (de) 1997-05-05 1998-02-25 Isolierung und Verwendung von Nukleinsäuremolekülen, welche für Mitglieder der SSX-Familie kodieren

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EP1005532A1 EP1005532A1 (de) 2000-06-07
EP1005532A4 EP1005532A4 (de) 2001-03-14
EP1005532B1 true EP1005532B1 (de) 2002-12-04

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EP98910056A Expired - Lifetime EP1005532B1 (de) 1997-05-05 1998-02-25 Isolierung und verwendung von nukleinsäuremolekülen, welche für mitglieder der ssx-familie kodieren
EP02024769A Expired - Lifetime EP1300463B1 (de) 1997-05-05 1998-02-25 Isolierung und Verwendung von Nukleinsäuremolekülen, welche für Mitglieder der SSX-Familie kodieren

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US (3) US6291658B1 (de)
EP (2) EP1005532B1 (de)
JP (1) JP2001527408A (de)
CN (1) CN1264424A (de)
AT (2) ATE229069T1 (de)
AU (1) AU729060B2 (de)
CA (1) CA2287902C (de)
DE (2) DE69828654T2 (de)
DK (1) DK1005532T3 (de)
ES (1) ES2187939T3 (de)
PT (1) PT1005532E (de)
WO (1) WO1998050528A1 (de)
ZA (1) ZA982009B (de)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6287756B1 (en) * 1997-05-05 2001-09-11 Ludwig Institute For Cancer Research Methods for determining presence of cancer in a sample by determining expression of an SSX gene
US7429639B2 (en) * 1999-09-29 2008-09-30 Ludwig Institute For Cancer Research SSX-2 peptides presented by HLA class II molecules
US7158977B2 (en) * 2003-11-21 2007-01-02 Lenovo (Singapore) Pte. Ltd. Method and system for identifying master profile information using client properties selected from group consisting of client location, user functionality description, automatically retrieving master profile using master profile location in autonomic computing environment without intervention from the user
US7858743B2 (en) * 2004-09-09 2010-12-28 Ludwig Institute For Cancer Research SSX-4 peptides presented by HLA class II molecules
US20110097449A1 (en) * 2006-06-30 2011-04-28 Conagra Foods Rdm, Inc. Seasoning and method for seasoning a food product while reducing dietary sodium intake
NZ575995A (en) * 2006-09-27 2011-03-31 Conagra Foods Rdm Inc Seasoning and method for enhancing and potentiating food flavor utilizing microencapsulation
CN102251035B (zh) * 2011-07-05 2013-01-23 北京大学人民医院 基于ssx-2基因辅助诊断多发性骨髓瘤患者的定量检测试剂盒

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* Cited by examiner, † Cited by third party
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GB9414580D0 (en) 1994-07-19 1994-09-07 Cancer Res Campaign Tech Materials and methods relating to the diagnosis of synovial sarcomas
US5888751A (en) * 1997-07-15 1999-03-30 Ludwig Institute For Cancer Research Method for diagnosis and treating cancers, and methods for identifying pathogenic markers in a sample of normal cells

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JP2001527408A (ja) 2001-12-25
CN1264424A (zh) 2000-08-23
US6339140B1 (en) 2002-01-15
ATE229069T1 (de) 2002-12-15
DE69809930D1 (de) 2003-01-16
AU729060B2 (en) 2001-01-25
EP1300463B1 (de) 2005-01-12
DE69828654T2 (de) 2005-12-29
EP1005532A1 (de) 2000-06-07
ES2187939T3 (es) 2003-06-16
EP1300463A1 (de) 2003-04-09
ATE286968T1 (de) 2005-01-15
WO1998050528A1 (en) 1998-11-12
ZA982009B (en) 1999-09-06
US20030023057A1 (en) 2003-01-30
EP1005532A4 (de) 2001-03-14
DK1005532T3 (da) 2003-03-17
DE69828654D1 (de) 2005-02-17
US6291658B1 (en) 2001-09-18
CA2287902C (en) 2007-04-17
DE69809930T2 (de) 2003-09-04
PT1005532E (pt) 2003-04-30
AU6439298A (en) 1998-11-27
CA2287902A1 (en) 1998-11-12

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